Art nucleotide segments, vectors, cell lines methods of preparation and use

ABSTRACT

A gene and gene product that regulates the expression of the capsidal envelope genes of HTLV-III/LAV and that can be used to regulate the expression of heterologous (non-viral) genes as well is disclosed. This art gene consists of two exons and can be used in creating nucleotide segments, vectors and cell lines. A new method for screening for compounds that inhibit the replication of HTLV-III is also described and comprises: 
     (1) transfecting a T-cell line with the HTLV-III art and env genes; 
     (2) thereafter, adding a preselected compound to the transformed cell line in increasing concentrations; and 
     (3) determining whether the compound effects the art function without being toxic to the cell. 
     An additional parameter to use in diagnosis of AIDS disease is also described. The use of the art gene and gene product in AIDS therapy is also disclosed.

The present invention is directed to the use of vectors, transformantsand cell lines containing the art gene, and the use of the art geneproduct for expression, diagnostic and therapeutic means. Moreparticularly, the art gene product can be used to regulate the rate ofexpression of heterologous gene products.

Considerable effort has been spent over the years in attempting tounderstand the mode of action of viruses, particularly that ofretroviruses. Questions for which answers have been sought include thereasons that certain of these viruses preferentially infect and/orreplicate in certain types of cells as opposed to other types of cellsand how the virus regulates its life cycle.

The Acquired Immune Deficiency Syndrome (AIDS), and AIDS-related complexhave been the subject of intensive scientific research and publicconcern. Human T-Cell leukemia virus III (HTLV-III)/LAV is theetiological agent of the acquired immune deficiency disease,AIDS-related complex and other virus-related disorders includingdegeneration of the central nervous system, lymphoid interstitalpneumonitis (LIP) an increased incidence of Kaposi's sarcoma, B-celllymphoma of a Burkitt's type, Hodgkin's lymphoma and thrombocytopenicpurpera, collectively called HTLV-III/LAV related disorders [F.Barre-Sinoussi et al., Science 200: 868(1983); R. C. Gallo et al.,ibid., 224: 500 (1984); J. Schupback et al., ibid.: 503; M. G.Sarngadharan et al., ibid.: 506; J. A. Levy et al., ibid.: 225, 840(1984); D. Klatzmann et al., Nature (London) 313: 767 (1984); M.Gottlieb et al., New England J. Med. 305: 1425 (1981); H. Masur et al.,ibid.: 1431 F. Siegal et al., ibid.: 1439; H. Lane et al., ibid.: 309,453 (1983); J. Ziegler et al., ibid.: 311, 565 (1984); G. Shaw et al.,Science 227: 177 (1985). D. Klatzman et al., Science 225: 54 (1984); M.Seligman et al., New England J. Med. 311: 1286 (1984)] AIDS isclinically typified by depletion of T-Cells of the T4⁺ (helper) subset,a phenomena reflected by cytotoxicity of the virus for T4⁺ cells invitro. Large scale production of the virus was made possible by thedevelopment of T4⁺ cell lines that were susceptible to virus infectionbut that were partially resistant to its cytophathic effects [M. Popovicet al., Science 224: 497 (1984).]

The HTLV-III genome, like that of other retroviruses, contains threeopen reading frames encoding the capsid proteins (the gag gene), theenvelope proteins (the env gene), and non-structural proteins necessaryfor replication (the pol gene) [Ratner, L. et al. Nature 313: 227-284(1985); Wain-Hobson, S., et al. Cell 40: 9-19 (1985); Sanchez-Pescador,R. et al. Science 227: 484-451 (1985); Muesing, M. A. et al., Nature313: 450-458 (1985); Robey, W. G. et al., Science 228: 593-596 (1985);Veronese, F. et al., Science 229: 1402-1405 (1985); Kitchen, L. et al.,Nature 312: 367-370 (1984); Schupbach, J. et al., Science 228: 503-505(1984); and Allan, J. S. et al., Science 228: 1091-1093 (1985)].

This genome also contains other open reading frames that encode at leastthree additional proteins not common to most retroviruses [Ratner L. etal., Nature, supra; Wain-Hobson, S. et al., Cell supra,Sanchez-Pescador, R. et al., Science, supra; Muesing, M. A. et al.,Nature supra; Arya, S. et al., Science 229: 69-74 (1985); Sodroski, J.et al., Science 229: 74-77 (1985)]. Mutations in two of these openreading frames (the sor gene that encodes a 23 kD protein [Sodroski, J.et al., Science 231: 1549-1553 (1986); Lee, T. H. et al., Science 231:1546-1549 (1986)] and the 3'orf gene that encodes a 27 kD protein,[Allan J. S. et al., Science 230: 810-812 (1985)]) do not eliminate theability of the virus to replicate in and to kill T lymphocytes[Sodroski, J. et al., Science 231: supra]. The transactivator(tat_(III)) gene encodes a 14 kD protein that post-transcriptionallystimulates HTLV-III long terminal repeat (LTR)-directed gene expression[U.S. patent application Ser. No. 806,263 filed Dec. 6, 1985; Rosen, C.A. et al., Nature 319: 555-559 (1986); Sodroski, J. G., et al., Science227: 171-173 (1985); Arya, S. et al., Science 229, supra; and Sodroski,J. et al; Science 229 supra which are incorporated herein by reference]via an interaction with specific target sequences (called TAR) in theleader of viral messages [Rosen, C. A. et al., Cell 41: 813-823 (1985)].Mutations in the 5' portion of the first coding exon of the bipartitetat_(III) gene destroy the ability of the virus to efficientlysynthesize structural proteins and to replicate [U.S. patent applicationSer. No. 806,263; Dayton, A. et al., Cell 44: 941-947 (1986)]. Thesemutations can be complemented in trans in cell lines that constitutivelyexpress the tat_(III) protein.

We previously discovered that it is possible to use the tat_(III) geneand gene product to produce high levels of heterologous gene products.However, the production of certain gene products such as envelopeprotein can result in lysis of the cell. Consequently, the cell will diebefore producing large amounts of the desired protein.

Further, some cells possess proteolytic enzymes that break downheterologous protein and prevent the accumulation of large amounts ofthe heterologous protein.

It would be desirable to have a system where large amounts of "buildingblocks", the messenger RNA (mRNA) species corresponding to specificproteins, of a desired protein could be accumulated in a cell beforeproduction of that desired protein began and to then initiateproduction.

It would also be desirable to have additional means of identifyingindividuals possessing the HTLV-III/LAV virus.

Further, it would be advantageous to have a new mode of findingcompounds that will prevent the infection, replication, propagation andspread from individual to individual of the cytopathic effects of theHTLV-III/LAV virus.

Still further, it would be beneficial to be able to produce non-lethalHTLV-III/LAV virus for both diagnostic and prophylatic purposes.

SUMMARY OF INVENTION

We have now discovered a gene and gene product that regulates theexpression of the capsidal envelope genes of HTLV-III/LAV and that canbe used to regulate the expression of heterologous (non-viral) genes aswell. This art gene consists of two exons and can be used in creatingnucleotide segments, vectors and cell lines. Additionally, we have foundthat this gene and gene product is necessary for the prolificreplication of HTLV-III/LAV. Thus, we have found a new method forscreening for compounds that inhibit the replication of HTLV-III. Thismethod includes the steps of:

(1) transfecting a T-cell line with the HTLV-III art and env genes;

(2) thereafter, adding a preselected compound to the transformed cellline in increasing concentrations; and

(3) determining whether the compound effects the art function withoutbeing toxic to the cell.

A variation of this method involves the establishment of cell lines thatcontain the art sequences integrated into the cellular DNA and expressart activity constitutively. Thereafter, steps 1 to 3 can be performed.

This gene and gene product can also be used in controlling theproduction of a desired heterologous gene product. This method includesthe steps of:

(1) transfecting a preselected cell line with a vector containing asufficient amount of the HTLV-III LTR to be responsive to atrans-activating protein upstream of a desired heterologous gene fusedto a cis-acting negative sequence, capable of releasing a cis-actinginhibitory factor; and

(2) at a predetermined time contacting the cis-acting inhibitory factorwith a sufficient amount of art gene product to repress the cis-actinginhibitory factor and permit expression of the desired heterologous geneproduct.

Further, this newly discovered protein of about 116 amino acidassociated with HTLV-III/LAV, results in an additional parameter to usein diagnosis of the disease. Still further, the art gene and geneproduct can be used in AIDS therapy. For this purpose purified artprotein or peptides derived therefrom, produced in bacteria, yeast ormammalian cells or synthesized chemically can be used to detectantibodies to the art protein in body fluids. Alternatively, antibodiesraised to the art protein or peptides derived therefrom can be used todetect art protein in tissues or body fluids.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the structure of the HTLV-III/LAV genome containing exonsof the art gene.

FIG. 1B shows the DNA sequence of the two open reading frames thatconstitute the art gene and the predicted amino acid sequence of the artgene product.

FIG. 1C shows the hydrophilic (upper)-hydrophobic (lower) profile of theart gene product.

FIG. 2A shows the structure of HTLV-III proviral deletion mutants.

FIG. 2B illustrates the replicative potential of the proviral deletionmutants as indicated by transfection into Jurkat-tat_(III) cells.

FIG. 3 (A-D) show the complementation of mutations in HTLV-IIIproviruses by plasmids designed to express the art gene product.

FIG. 4 is a schematic representation of plasmids containing the artgene.

FIG. 5A illustrates RNA slot-blots from transfected cells.

FIG. 5B shows proteins immunoprecipitated from the transfected cells.

FIG. 5C shows immunoprecipitates of cells transfected with proviralplasmids using patient antiserum.

FIG. 6 shows a plasmid that is capable of producing the art protein inbacteria.

FIG. 7 shows immunoprecipitates of the bacterially synthesized artprotein using a patient anti-serum.

FIG. 8 shows a plasmid in which a nonviral (heterologous) gene, in thisinstance chloramphenicol acetyltransferase (CAT), is negativelyregulated by cis-acting negative regulatory sequences present in the 3'terminal portion of the HTLV-III genome.

DETAILED DESCRIPTION OF THE INVENTION

We have now discovered a gene that produces a protein which in additionto the tat_(III) gene product, is necessary for efficient HTLV-III gagand env protein synthesis. The coding exons of this gene use alternativereading frames of the first and second coding exons of the tat_(III)gene (FIG. 1). Beginning at about a methionine codon at about nucleotide5550, the first coding exon of this gene extends to a known splice donorat about position 5625. The corresponding splice acceptor, located atabout nucleotide 7956, precedes an in-frame open reading frame ending ina stop codon at about position 8227. The splicing events needed toproduce this alternative reading frame product are the same as thoseused for the tat_(III) gene and occur in the messenger RNA of HTLV-IIIinfected cells.

The product of this alternative reading frame is about 116 amino acidslong and contains highly basic hydrophilic stretches similar to thosefound in the tat_(III) gene product and in nucleic acid-bindingproteins.

The expression of HTLV-III structural genes is governedpost-transcriptionally by the tat_(III) product, which acts as apositive regulatory factor, and by the art product, which counteractscis-acting negative-regulatory sequences located in or near the gag andenv genes. Two possible roles for this complex regulatory scheme can beconsidered. HTLV-III is reported to establish a latent state ofinfection in T-cells that are not activated [Zagury et al., Science 231:850-853 (1986)]. Lack of tat_(III) or art function would lead to a stateof infection characterized by accumulation of viral RNA withoutsynthesis of virus structural proteins. Rapid release form such a latentstate could be achieved in the absence of new RNA synthesis if thetat_(III) or art function were reconstituted. A dependence of tat_(III)and art activity on the state of cell differentiation would explain therelationship between HTLV-III latency and T-cell activation.

Alternatively, post-transcriptional regulators may play a part in thelytic cycle of the virus. An early stage of infection characterized byaccumulation of viral RNA but not of virion proteins might precede alate phase in which viral proteins toxic to T4 cells are produced. Theswitch from an early to a late stage of infection would reflectactivation of either one or both of the tat_(III) and art genefunctions. Such an early-late switch would permit accumulation of themRNAs for toxic virion components before such components themselves areproduced. There is some evidence that modulated tat_(III) activity mayresult in increased production of infectious virus. The yield of virusparticles on lytic infection of the Jurkat cell line that constitutivelyproduces high levels of the tat_(III) gene is much lower than thatobserved on infection of the Jurkat cell line itself, despite theobservation that the cytopathic effect of infection is accelerated inthe Jurkat tat_(III) cells. Premature cell death attributed to highconstitutive levels of the tat_(III) gene might explain the decreasedvirus titres. Temporal regulation of virus gene expression is importantin the life cycle of other lytic viruses. The postulated roles of thetat_(III) and art genes in the latent and lytic cycles of HTLV-IIIinfection are not necessarily exclusive.

The flexible multi-tiered regulatory pathway linked to host celldifferentiation and proliferation would account for much of thevariability observed in the disease consequent to HTLV-III infection[Seligman, M. et al., N. Eng. J. Med., 311: 1286-1292 (1984)]. Usingsuch a multi-tiered regulatory pathway involving both positive andnegative elements, should result in the production of high levels of adesired heterologous gene.

The necessity of the art gene product in addition to the tat_(III)protein for efficient HTLV-III gag and env protein synthesis isdemonstrated by preparing plasmids that delete parts of the two codingexons of the tat_(III) gene with the result that, the ability of thevirus to express gag and env proteins is dramatically reduced if noteliminated. Further, this attenuation of the virus is not the result ofdefective tat_(III) gene product, because these mutations are notcomplemented by the addition of tat_(III) gene products alone. Thus, itis clear that a second post-transcriptional control pathway is involvedin HTLV-III expression.

Raji cells that have been transfected with plasmids containing thesedeletions (pΔ(8053-8474 and pFS8053) failed to express HTLV-III gag orenv gene products, but did synthesize levels of the 14 kD tat_(III) geneproduct in amounts comparable to that seen following transfection withthe pHXBc2 plasmid. The pHXBc2 expresses the env, gag and tat_(III) geneproducts but does not synthesize a complete 3'orf gene product, asjudged by radioimmunopercipitation. Thus, mutations at or nearnucleotide position 8053 attenuate the ability of the provirus tosynthesize gag and env protein, but do not affect the synthesis of thetat_(III) protein. This is further shown in FIG. 2, which shows thatproviruses containing mutations in the 3' portion of the env gene areable to stimulate HTLV-III LTR directed gene expression to approximatelythe same level as is observed in the intact pHXBc2 provirus. Thus,mutations that effect gag and env gene protein expression do notsignificantly effect tat_(III) gene expression.

Significantly, plasmids which express the art gene product complementthe defect of the delection mutants and result in the synthesis of thegag and env gene product. HTLV-III LTR sequences from -167 to +80 wereplaced at various positions with respect to the putative initiationcodon for the art gene reading frame. Viral protein expression wasassessed at 48 hours following transfection.

FIG. 3 illustrates that placement of the HTLV-III LTR 54 nucleotides 5'to the art ACG codon (and downstream of the tat_(III) initiation codon)results in a plasmid (pEx5496) that provides in trans a functionrequired for gag expression by one of the mutant plasmids (pFS8053). Thegp160/120 env gene proteins are produced by the pEx5496 plasmid itselfupon transfection into Jurkat-tat_(III) cells. The ability of thepEx5496 plasmid to complement the pFS8053 mutation and to synthesizeenvelope proteins is eliminated by a frame shift mutation within thealternative reading frame (plasmid pEx5496FS). Plasmids in which theHTLV-III LTR is located 3' to the art ATG codon do not complement thepFS8053 mutations and also do not synthesize env gene products (Seeplasmids pEx5607 and pEx5702 in FIG. 4). In contrast, deletions withinthe env or 3'orf genes of plasmid pEx5496 do not eliminate the abilityto activate gag gene expression by the pFS8053 plasmid (See plasmidpEx5496Δ env and pEX(5496-8474) (FIGS. 3 and 4) Additionally, HTLV-IIIgag gene expression directed by the pΔ(8053-8474) plasmid can also beactivated by the pEx5496 plasmid. This shows that plasmids capable ofexpressing art gene products can activate in trans gag gene expressionby proviruses containing mutations that prevent expression of the artgene product.

We have further found that gag gene expression by the pFS8053 plasmidcan be activated in Jurket-tat_(III) cells by a plasmid that containsthe HTLV-III sequence located 5' to the initiation codon the tat_(III)gene. The pEx5365 plasmid produces functional tat_(III) activity, aswell as providing in trans the functions required for gag geneexpression (See FIG. 3). However, the extent of gag gene expressionobserved in this experiment was less than that observed in theexperiments in which the pEx5496 plasmid was used.

Plasmids designed to express the art gene product, but not the env genewere tested for the ability to allow env gene production by HTLV-IIIproviruses containing mutations for the art gene. The plasmidpEx5496Δenv (ATCC 67135) contains a deletion-frameshift mutation in theportion of the env gene encoding the exterior glycoprotein, gp120. FIG.3 illustrates that the pEx5496Δenv plasmid does not yield detectablelevels of the env protein upon transfection into Jurkat-tat_(III) cells.Co-transfection of this plasmid into Jurkat-tat_(III) cells with thepΔ(5365-5551) plasmid allows the latter plasmid to produce both gag andenv proteins (See FIG. 3). No viral protein could be detected inJurkat-tat_(III) cells transfected either with the pΔ(5365-5551) plasmidalone or with the pΔ(5365-5551) and pEx5496FS plasmids. The pEx5496Δenvplasmid also activates this synthesis of the gp160/120 env products bythe pEx5496FS and pEx5702 plasmids. No env gene products were detectedupon transfection of Jurkat-tat_(III) cells with the pEx5496 FS andpEx5702 plasmids alone. This demonstrates that the inability of theseplasmids to synthesize envelope protein is due to disruption of the artgene, rather than of cis-acting sequences required to produce envelopeprotein. This illustrates that a product expressed by the alternativereading frame, the art gene product, has a trans-activating functionwith respect to env gene expression and/or gag gene expression directedby proviruses mutated in the art gene.

Although, not wishing to be bound by theory, it is our belief that thereis a cis-acting negative regulatory sequence present on viral messagesencoding gag and env gene products which the art gene product depresses.This sequence results in a cis-acting inhibitory factor which preventsthe expression of the env and gag gene products. We have previouslyfound that the tat_(III) gene product alone is sufficient to stimulateHTLV-III LTR sequences (-167 to +80) to direct high level synthesis ofheterologous gene products such as bacterial chloramphenicolacetyltranferase and murine dihydrofolate reductase [(See U.S. Pat. No.806,263 filed Dec. 6, 1985; Rosen, C. A., Nature, supra, Sodroski, J.G., et al., Science 227: 171-173 (1985)]. However, plasmids containingthese same HTLV-III LTR sequences located 5' to the HTLV-III env gene(pEx5496FS or pEx5702) yield no detectable envelope protein in theabsence of the art gene product, even in the presence of an activetat_(III) gene product. It is only after this art gene product issupplied in trans do the levels of envelope production by the plasmidsapproximate those of the heterologous genes under the control of theHTLV-III LTR in Jurkat-tat_(III) cells. These observations indicate thatsequences in the pEx5496FS and pEx5702 plasmids 3' to +80 inhibit envgene expression, and that such inhibition can be relieved by the artgene trans-activating function. The requirement for this secondtransactivator for gag gene expression suggests that gag gene messagesalso contain repressor sequences in addition to those found in anv genemessages. It is for the above reason that we have chosen the name artfor the gene that encodes the second transactivator, standing foranti-repressor transactivator, consistent with the proposed role of thisgene in negating the function of cis-acting repressor sequences presenton viral messges encoding the HTLV-III structural proteins.

To test for the presence of a specific repressor sequence in the viralgenome the following recombinant plasmid was constructed (See FIG. 8).The bacterial chloramphenicol acetyltransferase (CAT) gene under controlof the HTLV-III LTR was deleted of its polyadenylation signals and wasjoined to the 3' end of the HTLV-III genome. Following transfection ofcells with this plasmid DNA CAT activity is virtually undetectable.However, co-transfection with an art expressor plasmid relievesrepression and CAT activity is increased. These experiments demonstratethat the HTLV-III genome contains a repressor sequence that can belinked to heterologous genes in order to control their activity. FIG. 8illustrates as described above that art gene activity can relieve thenegative effect of the HTLV-III cis-acting negative regulatorysequences. The sequences used for transcription initiation are derivedfrom the HTLV-III LTR in this experiment and there may be a specificinteraction between the HTLV-III LTR sequences and sequences in the gagand pol genes, respectively, required for the negative regulatory effectspecifically relieved by art functions.

Detectable levels of the tat_(III) gene but not of the gag and env geneproducts are synthesized by proviral mutants defective for the artfunction. The influences that inhibit gag and env gene products do notaffect tat_(III) gene expression. It is known that sequencesencompassing the entire gag gene and most of the env gene are removed bysplicing from the tat_(III) messenger RNA [Muesing, M. A. et al., Nature313, supra, Arya, S., Science 229, supra, Sodroski, Science 229, supra].Removal of art-responsive cis-acting repressing sequences located in theregions spliced out of tat_(III) messages, explain the independence oftat_(III) expression from the requirement from the art product. The artproduct should also be independent of such a negative regulatorysequence as it is synthesized from messages that also lack these gag andenv sequences. Heterogenity observed in the non-coding leader sequencesof potential tat_(III) and art messages could determine ATG usage, ineffect, modulating relative levels of tat_(III) and art proteins.

We have now found a new process for screening for a compound that willmitigate the cyopathic effects of the HTLV-III/LAV virus. This processinvolves screening for a drug that will inactivate the art gene product.As discussed above, we now know that for the HTLV-III/LAV virus toexpress the capsidal envelope proteins at any significant levels, theart gene product is necessary. Because the envelope structural proteinis necessary for the replication of the virus, by inactivating the artgene product and consequently preventing the replication of the envelopeprotein, it will be possible to mitigate, if not completely eliminate,the growth and the cytopathic effects of the virus.

The envelope protein kills T-cells in a very specific way. This proteinhooks onto the T4 receptor of T4 cells and fuses the cells together.Thereafter, the fused cells die. Thus, by introducing a vectorcontaining art gene and the env gene under the control of an HTLV-IIILTR into a T-cell line, one can assay for a compound that inactivatesthe art gene and consequently prevents expression of the envelope geneproduct and thus, stops the fusion. Preferably T-cells that areparticularly sensitive to the cytopathic effects of the envelope proteinare used. More preferably, T4 cells would be used.

Although, any T4 cells can be used, preferably cell lines derived fromHUT 78 cells, C8161 cells and Jurkat cells are used. More preferably,tat_(III) cell lines are used, as disclosed in U.S. patent application806,263. Most preferably, tat_(III) cell lines derived from C8161 cellsare used.

For example, cells of the C8166 T4⁺ lymphocyte line may be chosen asrecepients for the assay because this line expresses markers typical ofactivated T-cells and is equisitively sensitive to HTLV-III infectionand cytopathicity. Just before the C8166 cells show their maximumHTLV-III specific, positive membrane fluorescence and extracellularreverse transcriptase (RT) activity, cytopathic changes occur thatinclude syncytia formation, cellular enlargement, and extrusion of cellmembranes. The number of viable cells decrease rapidly thereafter. Nosuch changes occurred in C8166 cultures transfected with vectors thatwill not expresss the HTLV-III viral env gene.

Preferably, the cell lines will also contain a marker that is releasedupon the cell's death. This marker can be used to determine thecytopathic effect of the tested material. Thus, when a cell dies, themarker is released into the culture medium resulting in a reaction withthe medium that is visually observed. Such markers can be readilyselected by one of ordinary skill in the field and include, for example,chromium.

The assay system comprises transfecting a T4 cell line with theabove-described art-env vector. Thereafter, a compound is added to thecells that would be expected to inactivate the art gene product inincreasing dosages. Because the introduction of this vector wouldnormally be cytopathic to the cell, whether or not the compoundinactivates the art gene product is determined, merely by looking atwhether or not the transfected cells died.

Typically, after transfection with these vectors, the cells will showcytopathic changes. Usually, six days after transfection, there is adramatic decrease in the viability of the culture. Extracellular RTactivity is detected in cell-free supernatants of such cultures. Thecells demonstrate membrane fluorescence and die. Generally, this willoccur within two weeks after transfection. Consequently, if the celldoes not die, it can be assumed that the drug was effective ininactivating the art gene product. Further, if the compound being testedis cytopathic to the drug, it will kill the cell and release the markerin the cell. As an added control, one would preferably run parallelexperiments with T4 cells that are mock-transfected. Such cell linescould not be killed by expression of the envelope protein, and one wouldreadily be able to determine whether or not the compound being tested,and/or the concentration at which it is being tested is detrimental tothe viability of a cell.

Preferably the transfection occurs by cocultivation of the T4 cellcultures with an art cell line. This cell line would be prepared bytransfecting, for example, a B-lymphocyte cell line, such as Raji cells.The ability of these art cells to consitutively express the HTLV-III artand env products can be readily determined prior to cocultivation bytechniques well known in the art. Preferably, this cell line would alsobe able to express the tat_(III) protein. It is possible to establishstable cell lines that express these genes and therefore, one has aneasy and reliable method to transfect T4 cells each time one wants totest a new drug. For example, one could simply treat these art cellswith mitomycin C and cultivate the cells with T4 cells, such as C8166cells. The ratio of art cells to T4 cells can vary widely. Typically,the ratio ranges from 5 to 1 through 1 to 5, preferably, the ratio isabout 1 to 3. Thereafter, the T4 cells that have been cocultivated withthe art cells will show cytopathic changes, indistinguishable from thoseobserved after transfection of the T4 cells.

Preferred cell lines that express the art gene product include Raji,Hela, NIH 3T3, Jurkat, T-cell, B-cell and CHO.

It is preferable to screen compounds that prevent the interaction of theart protein with the sequences responsive to the art protein in theHTLV-III LTR or prevent the ability of the art protein to trans-activatethe HTLV-III LTR. HTLV-III structural gene expression is governedpost-transcriptionally by the tat_(III) product that acts as a positiveregulatory factor and by the art product that counteracts a cis-actinginhibitory factor resulting from cis-acting negative sequences locatedin or near the gag and env genes. Consequently, using compounds thatinhibit translation, such as substances that affect the formation oftranslational initiation complexes or alter the bonding of ribosomes tothe viral mRNA is most preferable.

Examples of compounds that can be used in this screening process includecompetitors, compounds that inhibit translation and compounds that alterthe binding ability of a compound. Compounds such as those described inthe Physicians' Desk Reference, 38th ed. Medical Economics Co., Droden,N.J. (1984), which can be used in the present screening process can bereadily determined by the person of ordinary skill in the art based uponthe above disclosure.

A preferred group of competitors would be mutant art proteins that wouldretain their ability to bind to nucleic acid but are deficient inovercoming the inhibitory affect of the cis-acting factor. Such proteinsshould serve as efficient competitors for functional art proteins.Random mutagenesis by, for example, chemical modification can be used togenerate large numbers of art mutants without a specified target region.In one embodiment, one would use the first coding exon of art which canbe isolated using convenient restriction endonuclease sites. This regionwill be cloned into the replicative form of phage M13.

Single stranded M13 containing the art insert in either orientation ismutagenized using methoxyalamine. This can generate single and doublenucleotide substitutions at a frequency of greater than 50% [(Kadonagaand Knowles, Nucl. Acids Res., 13: 1733 (1985)]. The single stranded DNAof a clone in one orientation is annealed to that of a clone in anotherorientation so that a double stranded insert is reconstituted. Thechemically modified inserts are removed from vector M13 DNA byrestriction digestion and recloned into cut alkaline-phosphatase treatedM13 replicative form DNA. Clones containing inserts are identified bythe colorless plaques generated when the insert disrupts thebeta-galactosidase gene present in the M13 vector. The insert fragmentcan then be sequenced using the dideoxy method of Sanger et al., PNAS,74: 5463: (1977)

Following generation and sequencing of art mutants in M13 by the methodsdescribed above, the insert fragments are recloned into an HTLV-IIIexpression vector containing HTLV-III LTR, and transfected intoeukaryotic cells. The activity of the mutant art proteins will bedetermined by testing their ability to activate the HTLV-III LTRdirected HTLV-III gag or env gene protein synthesis in cells that alsocontain the HTLV-III provirus intact except for a mutation thatinactivates the art gene for example the provirus on plasmid pFS8053 inco-transfection assays. Using T4 cells transfected with gag and env and,preferably also the tat_(III) gene, but not an unmodified wild type artgene, as many as 100 plasmid clones can be tested for activity in theperiod of one week. Moreover, mutations that increase or decrease thetrans-activating ability of the mutant art gene product can be detectedin a quantitative manner by looking at the degree and speed of celldeath.

Those mutants that are no longer able to trans-activate will be testedin the above-described screening process. If a mutant art protein thatcan effectively compete with the active form is found then the mutantart gene will be subcloned into the retroviral vector.

Art protein may also be used to test for the presence of HTLV-III/LAV.For example, art protein may be purified by reversed-phase HPLC and usedto elicit antibodies. This protein is then used to immunize rabbitsusing techniques well-known to the person of ordinary skill in thefield. Rabbit antiserum to art protein is then used in animmunoprecipitation analysis in potentially HTLV-III/LAV infected cells.Cells are metabolically labelled and the cell extract isimmunoprecipitated with the rabbit antiserum. Western blot assay istypically used [See Samuel, D. P. et al., Science 226: 1094 (1984)].Antigen-antibody complexes may be detected by known techniques, forexample, using a radioactive labeled protein. FIG. 7 indicates that theart protein is approximately 19 to 20 kilodaltons in molecular weightand elicits an immogenic response in infected patients. The availabilityof bacterially produced art protein will make it possible to carry outserological studies on the prevalance of antibodies during the diseasecourse. Besides its use as a diagnostic reagent, purified art proteinwill make it possible to fully study its biochemical properties.

The vectors used in the present invention can be in the form of plasmidsor viral vectors such as those described in PCT/US85/00986 filed May 24,1984. For example, the defective retroviral vector pZIPNEOSV(X)1prepared as described by Cepko et al., supra contains Moloney murineleukemia virus LTR's, polyadenylation signals, sequences required forreverse transcription and for encapsidation of RNA, as well as the 5'and 3' splicing signals that normally produce subgenomic env genemessenger RNA. This vector also contains the bacterial gene for neomycinresistance (neo) which confers a dominant selectable resistance to theantibiotic G418 is eukaryotic cells (Souther, P. J. et al., J. Mol.Appl. Genet. 1: 327-341 (1982)) so that art transfected cells canreadily be identified. Preferably, the vector can contain any elementsuch as antibiotic resistance, which will permit easy detection of atransfected cell.

The HTLV-III/LAV art gene used herein was obtained from infectiousproviral clone HXBc2 and encodes an HTLV-III/LAV associated trans-actingfactor, although it can readily be obtained from other HTLV-III/LAVsources.

Cell lines which stably express the art gene can be created by infectionusing a vector containing the art gene.

DNA is introduced into the psi/2 (ecotropic) and psi AM (amphotropic)cell lines by the calcium phosphate coprecipitation method (Wigler etal., Cell 16: 777-785 (1979). These lines constitutively produce themurine leukemia virus proteins but cannot package the viral transcripts(Cone, et al., P.N.A.S. 81: 6349-6353 (1984); Mann, et al., Cell 33:153-159 (1983)). Two days following transfection, cells are selectedwith the antibiotic G418 (400 g/ml for fibroblast lines and 700 g/ml forlymphocytes). G418 resistant clones are evident in 7 to 10 days.Insertion of the art exons does not interfere with splicing eventsrequired for transcription of the neo genes. G418-resistant psi 2 andpsi AM clones are isolated and the virus from clones producing greaterthan 10³ infectious units per ml are used to infect the test cells.(King et al., Science 228: 554-558 (1985)). Cells resistance to G418 areobserved subsequent to infection of the cell lines tested.

By substituting the Moloney LTR with other modified LTR's a tissuespecific expression vector can be obtained. The vectors are constructedusing a tissue specific enhancer(s) operatively positioned in the samesequence with a heterologous DNA segment corresponding to thepolypeptide of interest, as well as a stop codon and polyadenylationsequence downstream (3') from that gene. The vector should also containa replication origin.

The vector contains at least the segment of an enhancer which determinesthe tissue specificity of that enhancer, hereinafter referred to as the"tissue specificity determinant." The vector preferably contains acomplete viral enhancer, rather than just the tissue specificdeterminant from such an enhancer and preferably the tissue specificdeterminant is part of the complete enhancers.

The promoter contained in the vector can be any of the known promoterswhich function to permit expression of a desired product in the host ofchoice. Preferably the promoter is a viral promoter from the same classof virus as the enhancer. The preferred class of virus is retrovirus,and the preferred viruses for use in conjunction with the invention arethe Akv, SL3-3, and Friend viruses.

The term "tissue specific" as used in this disclosure and claims, meansthat the vector operates to produce a greater amount of desired productin the targeted tissue than it does in other tissues under normalculture conditions. Tissue specific vectors may produce 1.5 to 1,000 ormore times as much expression product in the target tissue as in othertissues. These tissue specific expression vectors are more fullydescribed in PCT/US85/00986 which is incorporated by reference.

The tissue specific determinant can be homologous, meaning it came fromthe same virus as the promoter, or heterologous, in which case it is notfrom the same virus as the promoter. Heterologous tissue specificdeterminants can be excised from other viral systems, or can besynthesized using known techniques. Tissue specific determinants whichare specific to the target tissue can be identified by assay techniques,where vectors encoding an indicator or marker compound, e.g.,chloramphenicol acetyl transferase (CAT), an indicator which can beeasily quantified as described below, to determine which vectors areeffective in the tissue.

If desired, enhancer(s) from tissue specific vectors can be compared inDNA sequence to the enhancers which are not specific to the targettissue to determine the DNA sequence of the tissue specific determinant.Thereafter, at least the tissue specific determinant, preferably theentire enhancer, may be utilized in the desired vector containing theart gene and the resulting tissue specific vectors utilized to expressthis gene product in the tissue of choice.

Various cell lines can differ in their ability to take up and expressthe transfected art DNA. For example, Raji cels, HUT 78 cells, Jurkatcells, HeLa cells and NIH 3T3 cells are useful. Human T-cells andB-cells, generally are very useful. Another useful method of achievingtransfection with the art DNA is to use cells infected with eitherHTLV-I or HTLV-II.

The present invention also permits the development of a multi-tieredgene expression system. For example, by placing a desired heterologousgene under the control of the responsive sequence of an HTLV-III LTR andthe cis-acting negative sequences that are located in or near either thegag or env gene, one can prevent the expression of the desiredheterologous gene until the cis-acting inhibitory effect is "counterbalanced" by the art gene product. The entire HTLV-III LTR region neednot be used in the vector, the HTLV-III TAR +1 to +80 sequence inaddition to functional promotor and enhancer sequences either ofHTLV-III LTR origin or of heterologous origin (for exampleenhancer-promotor of other retroviruses, DNA viruses, or cellular genes)should be sufficient. The cis-acting negative sequences are obtained byfusing the cis-acting negative sequences of the env and/or gag gene tothe desired heterologous gene and placing this downstream of at leastthe HTLV-III TAR sequence. The cis-acting negative sequence can beobtained by using the HTLV-III gag gene sequence. Alternatively, thecis-acting negative, sequence from the env gene could be used insteadof, or in addition to, the sequences from the gag gene. Most preferably,one would just use a nucleotide sequences coding for the cis-actinginhibitory region.

One could use an expression vector, of the type described above,containing, for example, an HTLV-III LTR sequence, downstream of thissequence is the desired heterologous gene lacking polyadenylationsequences. This region would be limited to the HTLV-III sequencescontaining sufficient nucleotides of the HTLV-III gag gene and/or theHTLV-III env gene to convey the cis-acting inhibitory effect, butexcluding sufficient nucleotides of the art gene to express functiongene product. Preferably, sufficient nucleotides of the tat_(III) geneto express functional tat_(III) gene product and/or viralpolyadenylation sequences are also present. Such a vector can readily beconstructed by one of ordinary skill in the art, (See for example, FIG.8).

Thereafter, this vector would be used to transfect a cell. If the vectorused does not contain the tat_(III) sequence, then preferably, this cellwould also be contacted with tat_(III) gene product. This contactingwith a tat_(III) gene product can be accomplished by a variety ofmethods. For example, this vector could be used on a tat_(III) cellline, or one could subsequently transfect the transformed cis-negativesequence containing cell with a tat_(III) gene or adding operabletat_(III) gene product to this cell. As a result of the presence of thecis-acting sequences, one would obtain large quantities of mRNA for thedesired heterologous gene, but the heterologous gene would not beexpressed until the cell was exposed to a sufficient amount of the artgene product to repress the cis-acting inhibitory factor.

This exposure to the art gene product could be accomplished by a varietyof mechanisms. For example, one could add the art gene product directlyto the cell's culture medium at a desired preselected time.Alternatively, one could create art cell lines where the art geneproduct expression is under the control of a secondary factor. Forexample, one could develop a cell line where art gene production istemperature dependant. Thus, until the temperature is raised to acertain point, that cell would not produce sufficient amounts of artgene product to counteract the cis-acting inhibitory factor. When usingsuch an art cell line, one would wait until a pre-determined time beforeraising the temperature of the cell. One could readily determine howlong it takes a particular cell line with a given culture medium and ata given temperature to produce a specific amount of mRNA for theheterologous gene product.

Another method could have the art gene under the control of somechemical factor, which is affected by the addition of some compound,such as a hormone. These types of cell lines can be readily developed byone of ordinary skill in the art using standard techniques. For example,the art gene could be placed 3' to the mouse mammary tumor virus longterminal repeat or the metallothionein promoter, which are responsive todexamethasone and heavy metals, respectively.

Alternatively, at a desired time, one could either transfect the cellwith a vector containing the art gene or cocultivate the transfectedcell with an art cell line. This will again result in the cells beingexposed to the art gene product.

Upon exposure to the art gene product, in sufficient amounts, theinhibitory effect of the cis-acting negative sequence would be overcomeand the desired protein would be expressed rapidly. Because high levelsof mRNA have already been built up, before expression begins, one canreadily obtain expression of the desired gene product on high levels ina short period of time. When this expression is carried out in thepresence of tat_(III) gene product, very high levels of proteinproduction result. Thus, problems encountered with the expression ofheterologous genes such as cell death or enzymatic attack on theheterologous protein can be minimized. For example, in the latter caseone would know when the vast majority of the desired protein was beingproduced and could use known techniques to inactivate the enzyme evenincluding killing the cell and then collecting the desired protein.

If a gag (and/or env)-desired heterologous gene product fusion proteinis created, one can obtain the desired protein by cleaving the fusionpolypeptide and separating the desired gene product from the otherconstituents by techniques well known to one of ordinary skill in theart, such as centrifugation, chromatography, etc.

When a fusion polypeptide, including the envelope gene is created, thetransfected cell line is preferably a cell line other than a T-cellline. For example, a B-cell line would be most preferable.

This system also permits research regarding the effect of a single geneon a cell. By introducing into a cell, the desired gene to be studiedunder the control of the cis-acting inhibitory factor one can turn thegene "on" and "off" as desired by introducing the art protein.

This system can also be adapted for use in multicellular organisms, forexample, with trangenic mice. One line of mice can be created containingthe preselected gene to be studied under the control of the cis-actingnegative sequences by using standard techniques. In this line of mice,the gene would be permanently shut off. Another transgenic line can becreated that has the art gene and will express the art protein. Thesetwo lines of mice are then mated and the effects of the gene can bestudied because the hybrid offsprings will contain both the preselectedgene and the art gene.

The present invention can also be used to create a live attenuatedvaccine. By using a provirus, in which the functional part of the artregion is deleted, the virus, although capable of infecting the cells,is not able to express the envelope protein, and therefore, cannot causethe disease. Because of the small size of the two art exons, thisattenuated virus used would closely resemble the complete virus.

The present invention is further illustrated by the following examples.These examples are provided to aid in understanding of the invention andare not to be construed as a limitation thereof.

EXAMPLE 1 Construction of Vector Used to Establish Art Cell Lines

The defective retroviral vector pZIPNEOSV(X) developed by Mulligan andcoworkers [Cepko, et al., Cell 37: 1053-1062 (1984)] was used toconstruct a vector for establishing stable art cell lines. This vectorcontains Moloney murine leukemia virus LTRs, polyadenylation signals,sequences required for reverse transcription and for encapsidation ofRNA as well as the 3' and 5' splicing that normally produce subgenomicRNA. In addition, the vector contains the bacterial neomycin (neo)resistance genes that confers a dominant selectable resistance to theantibiotic G418 in eukaryotic cells [Southern and Berg, J. Mol. Appl.Genet. 1: 327-341 (1982)]. The art gene of HTLV-III was obtained frominfectious proviral clone HXBc2 and encodes the HTLV-III/LAV associatedtrans-acting factor (Arya et al., Science 229: 69-73 (1985); Sodroski etal., Science 229: 74-77 (1985); Sodroski, J. et al., Science 231:1546-1549 (1986); Fisher, et al., Nature 316, 262-265 (1985)].

In all plasmids prepared (See FIG. 4), HTLV-III LTR Sequences from -167to +80 are positioned at the nucleotide denoted in the plasmid name.FIG. 4 shows the structure of the 3'half of the HTLV-III genome basedupon the sequence of Ratner et al., Nature 313, supra, including thepositions of the env gene, LTR, 3' orf gene, two tat_(III) coding exons,the two art coding exons (here depicted as solid black boxes). Theposition of a stop codon in the 3' orf gene of the parental pHXBc₂ usedis denoted by a vertical broken line. The zig-zag lines representsignals for polyadenylation and splicing derived from the simian virus40 early region [See Mulligan, R. C. et al., Nature 277: 108-114(1979)].

All plasmids were made by standard procedures using restriction andmodification enzymes according to manufacturer's suggestions. For theproviral deletion mutants, the numerals in the plasmid name correspondto the endpoints of the deletion. These deletion mutants can readily bemade by a person or ordinary skill in the art. The four nucleotideinsertion (4 base pair) in plasmid pEx54996FS was constructed bytreating a BamHl-digested provirus with the large fragment of DNApolymerase I in the presence of nucleotide triphosphates and religatingprior to transfection of E. coli. The pEx5496Z env plasmid wasconstructed by digesting the pEx(5496-8474) plasmid with the enzyme StuI, ligating to eight base pair Kpn I linkers, digesting to completionwith Kpn I and ligating prior to E. coli transfection.

FIG. 1 shows the structure of the HTLV-III art gene end-product. Theupper figure (1A) depicts the open reading frames in the HTLV-III genomebased on the sequence of Ratner et al., Nature 313, supra. The verticallines represent the position of stop codons. The open reading frames forthe tat_(III) env and art are noted. The positions of known spliceacceptors (SA) and donors (SD) (See, e.g. Muesing, M. A. et al Nature313 supra) as well as the BamHl site used for mutagenesis (position8053) are denoted. The putative initiator methionine codons for thetat_(III) gene and art are delineated beneath the figure.

The middle figure (1B) shows the DNA sequence of the two open readingframes that constitute the art gene with the positions of the expectedsplice donor (SD) and acceptor (SA) sequences noted. The predicted aminoacid sequence of the potential product of the open reading frame isprovided beneath the DNA sequence. If this splice donor and acceptor areused, the amino acid sequence encoded by the sequence near the splicesite would be LYQSNPPPNP.

The lower figure (1C) shows the hydrophilic (up)--hydrophobic (down)profile of the predicted art product based on the program of Hopp andWoods [Hopp, T. P. et al., P.N.A.S. 78: 3824-3825 (1981)]. Proteindomains specified by the first coding exon (I) are separated by avertical line from those specified by the second coding exon (II). Theamino acid sequence of a strongly hydrophilic, basic domain is shownbeneath the profile.

EXAMPLE 2 Transfection of Cell Lines with Art Vector

Jurkat-tat_(III) cells were transfected by the DEAE-dextran procedureusing ten micrograms of the proviral mutant to be complemented and tenmicrograms of the plasmid to be tested for ability to complement themutation. Forty-eight hours after transfection, cells were labelled with³⁵ S-cysteine and cell lysates were immunoprecipitated using an AIDSpatient serum RV119. [Lee, T. J. et al., Proc. Natl. Acad. Sci. U.S.A.81: 3856-3860 (1984)]. The positions of the gp160 and gp120 env proteinsand the p55, p24, and p17 gag gene products are denoted. Transfectedplasmids in FIG. 3A were pΔ(5365-5496) (lane 1), pEx5365 (lane 2),pEx5496 (lane 3), pEx5607 (lane 4), pEx5702 (lane 5) and pIIIβ-globin(lane 6). Transfected plasmids in FIG. 3B were pΔ(5365-5496) (lane 1),pFS8053 plus pEx5365 (lane 2), pFS8053 plus pEx5496 (lane 3), pFS8053plus pEx5607 (lane 4) pFS8053 plus pEx5702 (lane 5), and pFS 8053 alone(lane 6). Transfected plasmids in FIG. 3C were pEx(5496-8474) (lane 1),pEx5496Δ env (lane 2), pEx5496FS (lane 3), pFS8053 plus pEx5496 (lane4), pFS8053 plus pEx(5496-8474) (lane 5), pFS8053 plus pEx5496Δ env(lane 6), pFS8053 plus pEx5496FS (lane 7), pΔ(5365-5551) plugs pEx5496env (lane 8), pΔ(5365-5551) plus pEx5496FS (lane 9), pΔ(5365-5539) pluspEx5496Δ env (lane 10), and pΔ(5365-5539) plus pEx5496FS (lane 11).Transfected plasmids in FIG. 3D were pEx(5496-8474) (lane 1),pEx5496Δenv (lane 2), pEx5496FS (lane 3), pEx5496Δenv plus pEx5496FS(lane 4), and pEx5496Δenv plus pFS8053. The predicted env gene productsynthesized by the pEx5496FS plasmid is 47 amino acids shorter than thewild type HTLV-III envelope due to the frameshift mutation.

EXAMPLE 3 Preparation of Deletion Mutants

All deletion mutant plasmids were made by standard procedures usingrestriction and modification enzymes according to manufacturer'ssuggestions. The 4 base pair (4 bp) insertion in the pFS8053 plasmidresulted from treating a BamHl-digested provirus with the large fragmentof DNA polymerase I in the presence of nucleotide triphosphates andreligating prior to transfection of E. coli.

FIG. 2 shows the structure and properties of the HTLV-III proviralmutants. The complete HTLV-III provirus on plasmid pHXBc2 along withknown genes is shown in the upper left figure (2A). [See Fisher, A. C.et al., Nature 316, supra]. The dark boxes represent the two codingexons of the tat_(III) gene. The vertical broken line in the 3' orf generepresents a stop codon present in the pHXBc2 provirus [Sodroski, J. etal., Science, supra (1986)]. The scale beneath the viral genesrepresents kilobases. Numbers correspond to those by Ratner, where theRNA cap site is designated +1. For proviral deletion mutants, thenumerals in the plasmid name correspond to the endpoints of thedeletion.

Replicative potential of the proviruses was tested by transfection ofCsCl-banded DNA into Jurkat-tat_(III) cells [Rosen, C. A., et al., J.Virology 57: 379-384 (1986)] using the DEAE-dextran technique [Queen etal., Cell. 33, supra]. The values in the MIF and CPE columns representthe number of days following transfection that greater than 95%HTLV-III-specific membrane immunofluorescence and greater than 95%cytopathicity, respectively, were noted in a typical experiment. Thesevalues were assessed as previously described by Sodroski,. J. et al[Science (1986) supra]. The value ">30 days" indicates that not greaterthan 2 percent HTLV-III-related membrane immunofluorescence orcytopathic effect was observed in the cultures, even up to 30 daysfollowing transfection. Reverse transcriptase assays [See Rho, R. M. etal, Virology 112: 335-342 (1981)] of cell supernatants in these casesdid not rise above background during the observation period. ND=notdone. (See FIG. 2B).

Assessment of viral RNA production was performed as described below.Viral protein production was assessed by transfecting cells with 10micrograms plasmid DNA using the DEAE-dextran procedure and labellingwith ³⁵ S-cysteine at 48 to 72 hours post-transfection. Labelled celllysates were precipitated with patient antisera (RV119 forJurkat-tat_(III) cells and 38-1 for Raji cells) and assessed for gag,env and tat_(III) protein production on SDS-acrylamide gels [Lee, T. J.et al., P.N.A.S. 81: 3856-3860 (1984)]. A positive (+) value indicatesdetectable gag (p55, p38, p24 and/or p17), env (gp160/120) or tat_(III)(p14) bands, whereas a negative (-) value indicates no detectable levelof these proteins above background.

Trans-activating ability (TA) was assessed by co-transfecting 10micrograms of the proviral plasmid with 10 micrograms of plasmidpU3R-III, containing HTLV-III LTR sequences from -457 to +80 5' to thechloramphenicol acetyltransferase (CAT) gene, into Raji cells [Sodroski,J. et al., Science 225: 381-384 (1984); Gorman, C. M. et al., Mol. Cell.Biol. 2: 1044-1051 (1982)]. Forty-eight hours after transfection, celllysates were assayed for CAT enzyme activity as described. Numbersrepresent percentage conversion of chloramphenicol to acetylated formsin one hour using equivalent amounts of protein lysate in a typicalexperiment. No effect on CAT activity directed by the pSV₂ CAT plasmid,containing the SV40 early region promoter 5' to the CAT gene, wasobserved with any of the mutant proviruses tested.

EXAMPLE 4 RNA and protein production following transfection

Approximately 5×10⁷ Raji cells were transfected with 10 micrograms testplasmid DNA and 10 micrograms pSV₂ β-globin DNA using the DEAE-dextrantechnique [Queen et al, Cell 33: 741-748 (1983)]. Forty-eight hourspost-transfection half of the cells were labelled with ³⁵ S-cysteine andimmunoprecipitated with 38-1 patient antiserum as described [Lee, T. J.et al, P.N.A.S. (1986) supra]. The other half of the cells was used fortotal RNA isolation using the guanidine thiocyanate-CsCl method[Chirgwin, J. M. et al, Biochemistry 18: 5294-5299 (1979)]. Fivemicrograms of RNA was slot-blotted onto duplicate nitrocellulose filtersand ten micrograms were size-separated on formaldehyde gels andtransferred to nitrocellulose [Thomas, P., P.N.A.S. 77: 5201-5202(1980)] (See FIG. 5A). One slot-blot was hybridized to a probe derivedfrom the complete β-globin cDNA sequence (column a). The other slot-blot(column b) and the Northern blot (lower figure) was hybridized to aprobe made from a pooled collection of Bgl II internal proviralfragments derived from the pHXBc2 plasmid. Filters were washed asdescribed by Thomas, P.N.A.S., supra, prior to the autoradiography.Proteins immunoprecipitated from the Raji transfectants are shown inFIG. 5B. Transfected test plasmids for the Northern blot, slot blots,and protein gel are: (1) pHXBc2, (2) pΔ(8053-8474), (3) pFS8053, (4)pΔ(5365-5496) and (5) a plasmid, pCRl, containing an incomplete HTLV-Iprovirus. The control lanes in this figure (lanes 1 and 4) werepreviously published [Rosen, C. A., et al, Nature (1986) supra]. FIG. 5Cshows immunoprecipitates of Jurkat-tat_(III) cells transfected withproviral plasmids using patient antiserum RV119, as previouslydescribed. Transfected plasmids were: pHXBc2 (lanes 1 and 11),pΔ(5365-5496) (lane 2), pΔ (5365-5702) (lane 3), pΔ(8053-8474) (lane 4),pFS8053 (lane 5), pIIIβ-globin, containing a HTLV-III LTR 5' to rabbitβ-globin cDNA sequences (lanes 6 and 7), pΔ(6617-7198) (lane 8),pΔ(5365-5551) (lane 9), and pΔ(5365-5539) (lane 10).

EXAMPLE 5 Expression of The Art Gene Product

The coding region of art from an HTLV-III cDNA clone (derived frompCV4.3 HTLV-III cDNA clone [Arya et al, Science, supra]), as describedabove, was inserted into the BamHI site of an overexpression vector.Such vectors are readily available to a person of ordinary skill in theart. Indicated in FIG. 6 are the frames of two plasmids (clones 1.10 and6.1) constructed to express the art_(III) coding region. Expression ispromoted from the bacteriophage lambda P_(L) promoter. The P_(L)promoter is normally repressed by the lambda cI repressor gene to avoidany problems of lethality due to over expression of any protein duringcloning. To monitor expression off the P_(L) promoter, the P_(L)-art_(III) plasmid is re-introduced into bacterial strains(N99cI^(ts857)) that carry a prophage carrying a temperature sensitivemutation in its lambda cI gene. The temperature sensitive strains arethen induced at 42° C. to overexpress the art protein.

FIG. 7A shows that a protein, approximately 19 to 20 kilodaltons isinduced in strains by P_(L) -art_(III) at 42° C. Confirmation that thisis the art product is shown by use of strains containing a plasmid withan out-of-frame art sequence (Clone 12.1). Lanes 3 and 4 of FIG. 7Aindicate that this clone does not induce any protein of the samemolecular weight proving that the induced protein is expressed from theP_(L) -art_(III) plasmid.

FIG. 7B shows that the bacterially produced art product is recognized byAIDS patient sera. This demonstrates that the protein is made ininfected patients, and is immunogenic.

EXAMPLE 6 Preparation of Multi-tiered Expression System

An expression vector was prepared using standard techniques (See, e.g.,Maniatis, T., et al, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory 1982). This vector contained an HTLV-III LTR and thechloramphenicol acetyl transferase (CAT) gene, downstream of theHTLV-III LTR. The CAT gene was fused to a nucleotide sequence derivedfrom the HTLV-III genome, starting with the tat_(III) gene andcontinuing through the viral polyadenylation sequence, with the art geneexcised. This nucleotide sequence was prepared by the same procedures asdescribed in Example 3. A Jurkat tat_(III) cell was transfected by theDEAE-dextran procedure using ten micrograms of the vector. Forty-eighthours after transfection, cell lysates were prepared and tested for thepresence of the CAT protein. No CAT expression was detected.Subsequently, these cells were cotransfected with ten micrograms of theLTR-CAT plasmid and ten micrograms of an art expressing plasmid asdescribed above. Thereafter, CAT expression was detected confirming theability of the art gene to "turn on" cellular expression of aheterologous gene under the control of a cis-acting negative sequencesderived from the viral genome.

All the references discussed above are incorporated herein by reference.

It is evident that those skilled in the art, given the benefit of theforegoing disclosure, may make numerous other uses and modificationthereof, and departures from the specific embodiments described herein,without departing from the inventive concepts, and the present inventionis to be limited solely by this scope and spirit of the appended claims.

We claim:
 1. A DNA segment comprising a nucleotide sequence coding forproduction of the HTLV-III art protein but not containing a sufficientnumber of nucleotide sequences to code for a functional HTLV-III proteinselected from the group of HTLV-III proteins consisting of 3' orfprotein, gag protein, env protein and tat protein.
 2. The DNA segment ofclaim 1 comprising a sufficient number of nucleotides shown in FIG. 1Bcorresponding to a first nucleotide sequence between about 5500 to about5625 and a second nucleotide sequence between about 7956 and about 8227to express a poplypeptide having a trans-acting function.
 3. A DNAsegment according to claim 1, and having the nucleotide sequences 5550to 5625 and 7956 to 8227 as shown in FIG. 1B.
 4. The DNA segment ofclaim 1, which does not contain a sufficient number of nucleotidesequences to code for functional tat protein.
 5. The DNA segment ofclaim 4 which does not contain the tat initiation codon.
 6. A vectorcomprised of non-HTLV-III sequences and the coding sequences of theHTLV-III art gene but not a sufficient portion of the coding sequencesof at least one of the HTLV-III genes selected from the group consistingof 3'orf, gag, env and tat to express a functional gene product.
 7. Avector containing a sufficient amount of the HTLV-III art gene, to beable to express an HTLV-III art gene product that exhibitstrans-activating activity, a sufficient amount of the HTLV-III LTRresponsive to the HTLV-III art gene product for trans-activation and anenhancer upstream of the HTLV-III responsive segment but not asufficient amount of at least one gene selected from the groupconsisting of 3' orf, gag, env, and tat to express a functional geneproduct.
 8. The vector of claim 7 wherein the sufficient amount of theHTLV-III LTR is the HTLV-III TAR element.
 9. The vector of claim 6,which does not contain a sufficient portion of the coding sequences ofthe tat gene to express a functional tat gene product.
 10. The vector ofclaim 7, which does not contain a sufficient amount of the tat gene toexpress a functional tat gene product.
 11. The vector of claim 9 whichdoes not contain the tat initiation codon.
 12. The vector of claim 10,which does not contain the tat initiation codon.
 13. A stable cell linecontaining and expressing the HTLV-III art gene but not containing asufficient number of nucleotides to express a HTLV-III protein-selectedfrom the group consisting of 3'orf protein, gap protein, env protein andtat protein.
 14. The stable cell line of claim 13, which does notexpress the tat protein.
 15. The stable cell line of claim 14, whichdoes not contain the tat initiation codon.
 16. The art cell line ofclaim 13 where the cell is a Raji cell line.
 17. The art cell of claim13 where the cell is a HeLa cell line.
 18. The art cell line of claim 13where the cell is a NIH 3T3 cell line.
 19. The art cell line of claim 13where the cell is a Jurkat cell line.
 20. The art cell line of claim 13where the cell is a human T-cell line.
 21. The art cell line of claim 13where the cell is a human B-cell line.
 22. The art cell line of claim 13where the cell is a CHO cell line.